Enzyme electrodes are based on coupling the reaction of the substrate to be determined under the action of immobilized enzymes with the electrochemical indication of an electrode-active partner of the enzyme reaction While enzyme reactions generally have a high selectivity, the electrochemical indication reaction, coupled to these reactions, generally is relatively nonspecific. The cause of this nonspecificity lies in the electrochemical reaction of all components of the test sample with a conversion potential below the potential of the indicator electrode. These interferences are excluded by permselective membranes, which are disposed either directly before the indicator electrode (U.S. Pat. No. 3,979,274) or between the test solution and the enzyme layer (Toshiba Rev. 132). These membranes have a selective permeability because of the pore exclusion volume or the charge on the permeant. Nevertheless, molecules with a molecular weight similar to that of the substances indicated at the electrode can lead to interference. Moreover the manufacture of such permselective membranes is expensive and the use in enzyme electrodes is inappropriate because of mechanical instability.
A different principle of excluding these interferences is based on measuring the difference between an enzyme-free indicator electrode an enzyme-loaded indicator electrode (German Federal Republic Pat. No. 1,598 285). Admittedly, this method permits the exclusion of the effect of interfering substances of the measurement sample. However, it requires very expensive equipment and frequent calibrations. Moreover, the method is unsuitable for "kinetic methods of measuring", since the kinetic behavior of these two different electrodes is different.
It has furthermore been proposed that the interfering substances of the test sample be removed by means of an enzymatic reaction in an additional layer before the actual enzyme layer. This method requires a specific enzyme for each interfering substance and is therefore suitable for use only in those test samples, which always contain the same interfering substances.
To achieve correct measurements with a laminated enzyme membrane, the enzyme layer was disposed between two semipermeable membranes. Although all species up to a molecular weight of 15,000 Dalton can pass through this laminated membrane, low concentrations of interfering substances do not cause any falsification of the values measured, since the H.sub.2 O.sub.2 formed in the enzyme reaction permeates significantly more rapidly through the enzyme layer and the second dialysis membrane. On the other hand, when determining urine glucose where the concentration of the interfering substances may be appreciably higher than that of the substances being analyzed, excessively high measurement values occur frequently.
Moreover, a bienzyme electrode is known (German Democratic Republic Pat. No. 236 553), which contains an enzyme (indicator enzyme), which converts the substance to be determined into an electroactive product, as well as an enzyme (eliminator enzyme), which converts the interfering substances into substances which do not interfere electrochemically. Interfering substances, which are not converted effectively by the eliminator system can be converted in the measurement solution by an upstream chemical reaction into substances, which do not interfere, and an interfering substance, which is a substrate of the eliminator enzymes. The effect of interfering substances was partially suppressed even by an additional eliminator electrode. The difference measurement and the use of an eliminator electrode require additional expense for equipment and are therefore appreciably more expensive than simple measurements. Moreover, the eliminator electrode does not lead to the complete exclusion of interference. The membranes described are also very expensive and, at the same time, very thin and not very stable.
The measurements are slowed down significantly through the use of several superimposed enzyme layers For this reason, they are not suitable for use in the conventional analyzers.
Furthermore, the functional stability of the eliminator enzyme frequently is decreased drastically by products of the indicator enzyme reaction, such as H.sub.2 O.sub.2.